Genes encoding MCM proteins were originally reported from budding and fission yeast. Eukaryotic MCM sequences reported so far are homologous to any of the six MCM genes identified in yeast. Recently MCM proteins were reported in Archaea genomes as well, pointing to their ancient existence and critical role in several organisms.
MCM2-3-5 proteins are nuclear-localized in yeast and are required for DNA replication from specific replication origins. The MCM mutants exhibit defects in cell cycle progression in yeast. In Drosophila, mutations in MCM2 or MCM4 (dpa) inhibit cell proliferation and are lethal, with mutants showing prolonged S phase. In Arabidopsis following prolifera (PRL) gene disruption by transposon mutagenesis, semi-sterile plants with reduced megagametophyte viability were observed. Thus a clear role for the prolifera gene was established in plants in megagametophyte and embryo development.
At least in yeast, Xenopus, and mammals, there is a considerable amount of literature available about the MCM proteins and some models about their specific role at the molecular level in DNA replication. DNA replication is a very complex and tightly regulated process in eukaryotes and the machinery has been documented to be broadly conserved across eukaryotes. In eukaryotes, DNA replication actually is initiated from several replication origins and by some controlling mechanisms, that apparently involve other proteins, the replication process is restricted to a single round per each cell cycle. This ensures that cells do not end up with multiple sets of genomes resulting in polyploidy. Initiation of DNA replication follows the formation of a complex of proteins called Origin Recognition Complex (ORC) which may probably permit loading of other required proteins for the process such as cdc6 in yeast (cdc18 in S. pombe). Overexpression of cdc18 in S. pombe induces multiple rounds of S phase in the absence of mitosis and thus cdc6/cdc18 together ensure one round of DNA replication per cycle. Mutants of MCM proteins suggest that these proteins bind to chromatin in G1 phase and disassociate in S phase through the end of mitosis. This phase-specific association ensures that the replication origins remain competent only at the end of G1 phase and fire only once during S phase leading to one cycle of replication. In general, binding of MCM proteins to chromatin also require other proteins such as cdc6/cdc18 in conjunction possibly with some protein kinases cdk2/cdc2 and cdc7-dbf4 regulate firing of origins of replication.
Cell division plays a crucial role during all phases of plant development. The continuation of organogenesis and growth responses to a changing environment requires precise spatial, temporal and developmental regulation of cell division activity in meristems (and in cells with the capability to form new meristems, such as in lateral root formation). Such control of cell division is also important in organs themselves (i.e. separate from meristems per se), for example, in leaf expansion, secondary growth, and endoreduplication. It has been observed that endoreduplication occurs in about 20% of leaf epidermal cells in maize and in trichomes. In endosperm, endoreduplication has been well documented in maize. An important feature of cell differentiation in maize endosperm is nuclear enlargement through chromosome endoreduplication. This process may be related to many differentiation events, such as protein and starch synthesis and storage, accumulation of nucleotides, enzyme activation, and hormone synthesis, and consists of subsequent cycles of DNA replication without entering mitosis until high ploidy levels are attained. Variations in chromosome endoreduplication frequency in endosperm parenchyma have been described among maize populations.
What is needed in the art are methods and compositions to facilitate multiple rounds of DNA replication without nuclear division leading to polyploidy. Increasing the frequency of endoreduplication in the endosperm increases the size of endosperm cells and thus the endosperm size. Additionally, initiation of endoreduplication in leaf and stem cells, particularly in maize, leads to increased biomass and vegetative growth which has advantages in increasing plant yield. The present invention provides this and other advantages.